Crystallins are water-soluble proteins that are highly refractive and are related to metabolic enzymes and stress-protective proteins. Crystallins are the dominant structural components of the vertebrate eye lens and can comprise up to 90% of the protein content. The evolutionary relationships of the three classes of crystallins (α, β and γ) present in mammals have been clearly established.
There are two α crystallin genes, αA and αB (for acidic and basic, respectively), encoding proteins that share approximately 60% sequence identity. αA and αB crystallins have two domains, a crystallin domain and an alpha-crystallin-HSP domain. Two other domains share homology to the alpha-crystallin-HSP domain, namely the HSP20 domain and IbpA domain. Alpha crystallins can be induced by heat shock and are members of the small heat shock protein (sHSP) family. They act as molecular chaperones and hold unfolded or misfolded proteins in large, water soluble low molecular weight aggregates. These heterogeneous aggregates consist of 30-40 subunits of alpha crystallins in which the αA and αB subunits are present in a 3:1 ratio.
Alpha crystallins are present in all animal kingdoms but not in all organisms. Only αB crystallin has been found to be stress inducible. The expression of αA crystallin is essentially limited to the eye lens with only traces found in some other tissues. As such, αA crystallin is an essentially eye lens specific member of the family. αB crystallin is more widely expressed and is particularly abundant in brain, heart and muscle (Bloemendal et al., 2004).
β crystallins are members of the beta/gamma-crystallin family. There are at least 5 different proteins comprising the β crystallins. The beta/gamma-crystallin family of proteins contains a two-domain β-structure, folded into four very similar “Greek Key” motifs. β crystallins form homo/heterodimer, or complexes of higher order. The structure of β-crystallin oligomers appears to be stabilized through interactions between their N-terminal arms. βB2 crystallin contains a duplication of the XTALbg domain. At least 5 gamma crystallins have been identified in bovine and rat lens.
α, β and γ crystallins are the major protein components of the vertebrate eye lens with alpha crystallin being both a molecular chaperone as well as a structural protein, whilst beta and gamma crystallins are structural proteins (Bloemendal et al., 2004). Lenticular proteins, such as the abundant water-soluble crystallins cannot be replaced and thus must last the lifetime of the organism. βB2 crystallin has been demonstrated as being essential for maintaining the high solubility of crystallins in the eye lens. Its expression does not appear to be induced in other tissues upon changes in physiological condition that occur during wounding.
Proteins can be considered as evolutionarily related when conspicuous sequence similarities can be detected over longer and contiguous stretches of residues. Such homologous proteins are accordingly grouped in families and superfamilies, with higher or lower than about 50% sequence identity, respectively. Notably, there are no close structural relationships between α crystallins and the β/γ crystallins with respect to domain structure or sequence homology.
Historically, crystallins have been classified into α, β and γ classes by the size of oligmers formed that correspond with the classes now identifiable through analysis of the respective gene sequences. Whilst alpha crystallin aggregates range from 600 to 180-80 kDa and beta crystallin aggregates range from 200 to 50 kDa, the gamma crystallins are monomeric and their relative molecular mass ranges from 20 to 25 kDa (Ajaz et al., 1997; Hejtmancik et al., 1997). Beta crystallins are the most varied in aggregate size, forming several distinct classes of aggregates: β H (primarily octamers of 160-200 kDa), β L1 (primarily tetramers of 70-100 kDa and β L2 (primarily dimers 46-50 kDa) (Hejtmancik et al., 1997).
Two α crystallins αA (acidic) and αB (basic) have been described as indicated above, whereas up to 7β crystallins are known; 3 basic—B1, B2, and B3; 3 acidic—A2, A3 and A4; and the seventh form called βS. Similarly, there are several gamma crystallins (A, B, D, E, and F). Other crystallins are also known such as zeta, lambda and the heat shock proteins (hsp) hspB1 and hspB8.
The αA, βB2 and βB3 crystallins can be phosphorylated at their penultimate serine residue. Many other post-translation modifications (PTM) are also known to occur in crystallin proteins that accumulate over time as these proteins are maintained throughout the life of the animal. The PTM identified include methylation, acetylation, phosphorylation, oxidation of tyrosine and tryptophan residues, glycosylation, glutathione, and S-methylcysteine covalent attachment. A review of the types of PTM that occur in lens crystallins is provided by Hoehenwarter et al., (2006).
β crystallins are abundant lens proteins in most, if not all, vertebrate species, and have been reported in chick non-lens tissues, both ocular and extra-ocular, including the expression of βB2-crystallin in the retina (Head et al., 1991). In addition, extralenticular β crystallin expression is found in mammals and βB2 crystallin has been shown to be expressed in mouse and cat neural and pigmented retinas as well as in cat iris (Dirks et al., 1998). Although present at levels lower than those found in the lens, the appearance and accumulation of βB2 crystallin in the neural retina coincides with the functional maturation of this tissue (Head et al., 1995). βB2 expression has also been reported in bovine testes and rat brain (Magabo et al., 2000).
Extralenticular expression of βB2 indicates that it may play a metabolic role in non-lens tissues in addition to its structural role in the lens. Consistent with a possible metabolic function, βB2 is phosphorylated by cAMP-dependent kinase (PKA). Surprisingly, two forms of the protein are detected by SDS-PAGE, only one of which is phosphorylated by PKA (Kantorow and Horwitz, 1997). Incubation of recombinant mouse βB2 and bovine βB2 under identical conditions without cAMP or PKA also resulted in phosphorylation. This in vitro autophosphorylation is dependent on Mg2+ and is serine specific. It has been shown that deletion of the βB2 C-terminal arm does not abolish autophosphorylation activity suggesting that autophosphorylation involves a different serine than the penultimate C-terminal serine identified for PKA phosphorylation.
Earlier reports identified and named a phosphorylated β crystallin protein in the eye lens as βBp (Kleiman et al., 1998). This protein was later renamed as βB2 crystallin and is synthesized in new cortical cells. The βB2 crystallin of the lens nucleus was shown to be decreased significantly in both absolute concentration and in its proportion of the total soluble protein fraction (McFall-Ngai et al., 1986). Post-translational changes in the nuclear soluble βB2 crystallin resulted in a gradual loss of approximately 3000 daltons in the apparent mass of the βBp molecule resulting in a 23 kDa protein which correlates with the relative molecular mass reported for βB2 crystallin.
International Patent Application No. PCT/US2004/014920 (WO 2005/004894) describes the treatment of conditions characterized by cell or tissue damage involving the administration of “protective proteins”. Protective proteins are defined in the application as being small molecular chaperone proteins such as αA crystallin, αB crystallin, γD crystallin, Sic A, p26, and high heat stable crystallins, that improve solubility and/or stability of proteins. P26 is a low molecular weight chaperone protein of encrysted brine shrimp. SicA is another member of the small heat shock protein family and is a chaperone protein of the type III excretion system of Salmonella. These chaperone proteins are stated to act by protecting other proteins from damage and function by minimize degradation from conformational changes and enzymatic cleavage or digestion, thereby enhancing cell and tissue viability. The properties of the protective proteins are stated to include enhancing wound healing.
International Patent Application No. PCT/JP2004/00609 (WO 2004/096277) relates to the use of a preventative or therapeutic agent for intraocular vascularisation disease. The agent is stated to be a crystallin inhibitory substance, and can comprise an antisense RNA oligonucleotide having a nucleotide sequence complementary to a sequence coding for βB2 crystallin. In particular, an animal model is described in which βB2 crystallin is expressed in or around retinal vasculature induced with exposure of the retina to high levels of oxygen. High oxygen conditions are important for propyl hydroxylase activity to form hydroxyproline in collagen, and the observation of the expression of βB2 crystallin is consistent with a structural role for the protein.
Any break in the skin, regardless of the cause gives bodily access to foreign pathogens, providing a fertile breeding ground and a potential site for serious infection that may become life threatening. Both acute and non-healing chronic wounds remain a challenge in terms of both treatment and to the health care system. As such, there is an ongoing need for improved treatments for acute and chronic wounds which often require intensive and costly treatments.